water

Article Bio-Removal of Methylene Blue from Aqueous Solution by Galactomyces geotrichum KL20A

Margarita Contreras 1, Carlos David Grande-Tovar 1,* , William Vallejo 1 and Clemencia Chaves-López 2

1 Grupo de Fotoquímica y Fotobiología, Universidad del Atlántico, Puerto Colombia 81007, Colombia; [email protected] (M.C.); [email protected] (W.V.) 2 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Via R. Balzarini 1, 64100 Teramo, Italy; [email protected] * Correspondence: [email protected]; Tel.: +57-5-3599484

 Received: 23 October 2018; Accepted: 7 January 2019; Published: 6 February 2019 

Abstract: The conventional treatments used to remove dyes produced as a result of different industrial activities are not completely effective. At times, some toxic by-products are generated, affecting aquatic ecosystems. In this article, an efficient use of microorganisms is presented as a biodegradation technique that is a safe environmental alternative for the benefit of aquatic life. A strain of the Galactomyces geotrichum KL20A isolated from Kumis (a Colombian natural fermented milk) was used for Methylene Blue (MB) bioremoval. Two parameters of the bioremediation process were studied at three different levels: initial dye concentration and growth temperature. The maximum time of MB exposure to the yeast was 48 h. Finally, a pseudo-first-order model was used to simulate the kinetics of the process. The removal percentages of MB, by action of G. geotrichum KL20A were greater than 70% under the best operating conditions and in addition, the kinetic simulation of the experimental results indicated that the constant rate of the process was 2.2 × 10-2 h−1 with a half time for biotransformation of 31.2 h. The cytotoxicity test based on the hemolytic reaction indicated that by-products obtained after the bioremoval process reached a much lower percentage of hemolysis (22%) compared to the hemolytic activity of the negative control (100%). All of these results suggest that the strain has the capacity to remove significant amounts of MB from wastewater effluents.

Keywords: bioremediation; dyes; Galactomyces geotrichum; methylene blue

1. Introduction Most of the industrial effluents in developed countries have a high load of xenobiotic compounds, such as dyes. These compounds are recalcitrant, difficult to biodegrade, and toxic to aquatic , animals, and human beings. Most of the dyes used in the textile industry are stable, resistant to biological, physical, and chemical treatments, implying that their complete removal is difficult. Due to their high solubility in water, they cause contamination in local aquatic systems when they are discharged directly without prior treatment [1]. In particular, Azo dyes are the largest synthetic chemical class of dyes that are characterized by the presence of one or more azo bonds (-N=N-). They are used to a great degree in textile, pharmaceutical, leather, food, cosmetic, painting, and large-scale printing industries due to their ease of synthesis and stability [2]. MB is an aromatic heterocyclic used in different applications (e.g., medicine [3], biological [4], and textile industries [5]). Currently, MB dye is one of the most commonly used substances for dyeing cotton, wood, and silk [6]. The dye is commonly found in industrial wastewater, which causes devastating effects on the environment [7]. Given the characteristics of solubility and stability of azo dyes, traditional methods of flocculation, sedimentation, or adsorption are not efficient in the removal of these compounds because they only

Water 2019, 11, 282; doi:10.3390/w11020282 www.mdpi.com/journal/water Water 2019, 11, 282 2 of 13 transfer matter between different phases without effective degradation of the compounds [8]. Currently, such physicochemical methods are the most important ones used for the treatment of wastewater produced by the textile industry, resulting in effluents of acceptable quality, while at the same time generating waste products with high levels of toxicity. For this reason, research on the application of biological processes as a method of dye degradation has been intensified in recent years due to the resulting low toxicity. Their high efficiency [9,10], which is due in part to the inherent capacity of the microorganisms to break down organic pollutants by using them as a source of carbon and energy, or by co-metabolism [11,12], has also contributed to intensified research. The use of for the bioremoval of toxic compounds has many advantages in comparison to filamentous bacteria and fungi, since they grow rapidly and have the capacity to adapt and resist unfavorable environments [13]. The Geotrichum spp. is a filamentous (Phylum , Order ) that has transparent spores by segmentation of vegetative filaments [14]. G. geotrichum is a holomorphic organism, which means that it presents two forms of reproduction (sexual and asexual): the asexual form of multiplication gives rise to the anamorph () or imperfect state, while the sexual form gives rise to the teleomorph (G. geotrichum) or perfect state. These fungal species are capable of alternating a unicellular phase (yeast growth) with another mycelium (hyphae) in response to changes in various environmental factors (nutrients, CO2 pressure, pH changes, temperature). They are called “dimorphic fungi” and tend to proliferate as yeasts in tissues. However, they assume filamentous forms at room temperature in the environment [15]. This fungus is present in diverse habitats. It is found as a component of the natural flora in milk and is used as a ripening agent for cheese. Furthermore, it presents colonies of fast growth (four days of maturation), and of unlimited size. G. geotrichum is white and has a moist, yeast-like, appearance, and is easy to obtain. This yeast is characterized by true hyphae, thick (macrosiphonated) forming numerous arthroconidia, hyaline, and rectangular. The absence of blastoconidia differentiates it from the spp. [16]. Their lipases and proteases produce fatty acids and peptides that can be metabolized by other microbial populations, promoting the development of different aromas [17]. G. geotrichum can be considered one of the fungi with a large number of biotechnology applications. In fact, 38% of the publications which refer to this organism mention its capacity to biodegrade toxic compounds and its application in bioremediation processes in the treatment of wastewater for the improvement of the environment [18]. For G. geotrichum MTCC (registered at Microbial Type Culture Collection and Gene Bank, MTCC) a 100% efficiency in methyl red discoloration was reported after one hour at 30 ◦C. The composition of malt yeast medium used for decolorization studies was malt extract (3.0 g/L), yeast extract (3.0 g/L), peptone (5.0 g/L), and glucose (10.0 g/L) (pH 7.0) [19], while Waghmode et al. reported the decolorization of the azo dye Rubine by G. geotrichum MTCC 1360 within 96 h from exposure at 30 ◦C and pH 7.0 [20]. Govindwar et al. also reported a reduction of 86% via discoloration of the RY-84A dye at 50 mg L−1 after 30 h of exposure [21]. Despite extensive studies on different microorganisms for the development of biotechnological applications, this is the first time that removal of MB with G. geotrichum KL20A has been reported. In this work, the removal of MB dye with G. geotrichum KL20A was studied in order to search for alternative methods of water treatment that would be environmentally friendly and safe.

2. Materials and Methods

2.1. Yeast Isolation and Culture The yeast G. geotrichum KL20A was isolated from traditional Kumis samples collected in Valle del Cauca (in the southwest of Colombia) as reported by Chaves-López et al. [22]. The identification was carried out by sequencing of the D1/D2 region of the 26S rRNA gene and by random amplification of polymorphic DNA (RAPD-PCR). Subsequently, it was sequenced and compared with the data available in The European Molecular Biology Laboratory (EMBL) nucleotide sequence database. The strain was maintained in Petri dishes containing 12 mL of YPD-agar medium (10 g/L yeast extract, Water 2019, 11, 282 3 of 13

20 g/L dextrose, 20 g/L peptone, 20 g/L agar) and periodically reactivated for 24 h at 30 ◦C in the same growth medium [19,21]. A suspension cell solution was then prepared in YPD-broth (without agar) and a chamber count was performed using a Neubauer chamber to control the concentration of the yeast (approximately 3.2 × 107 cel/mL) to be used in the removal tests. The Neubauer chamber remains the most common method for cell counting around the world, also known as hemocytometer.

2.2. Removal Tests

2.2.1. Effects of the Methylene Blue Concentration and Temperature Methylene Blue dye (MB CI 52015 from Merck KGa, Darmstadt, Germany) was used to prepare a stock solution of 3.13 M (1000 ppm, pH 7.3) by dissolving the required amount of the dye in sterile distilled water. Concentrations of 50, 100, and 200 ppm were used for the removal tests. To this end, sterilized tubes containing 1 mL of YPD-broth medium (pH 7.0) were added to the appropriate MB concentrations (50 ppm, 100 ppm, and 200 ppm) and inoculated with 3.2 × 107 cells/mL of G. geotrichum KL20A. Decolorization of MB was studied at 25 ◦C, 30 ◦C and 35 ◦C for 48 h. Periodically, 160 µL of the samples were transferred into a 96 well microplate and the absorbance at 660 nm was read using Elisa SPR-960 reader [23]. The removal tests were carried out in triplicate with their respective targets, under the strictest biosecurity conditions to preserve the asepsis of the experiments.

2.2.2. Effects of the pH In order to study the effect of the pH on the discoloration of the MB, we performed the experiment using the optimal conditions of concentration and temperature to degrade MB (50 ppm, 35 ◦C). Thus, we modified the above-mentioned pH of the medium to achieve values of 5, 7, and 9 using HCl/NaOH 0.1 N solutions. The pH was measured with a Thermo-Fisher Scientific pH meter, previously calibrated with buffer solutions of pH 4, 7, and 10. Also in this pH study, the inoculum was yeast suspension containing 3.2 × 107 cells/mL. Finally, biodegradation kinetics were investigated during the 48 h and a pseudo-first-order model was used to simulate the data and derive the biodegradation rate constant.

2.3. Citotoxicity Analysis The evaluation of the toxicological impact of the MB dye and its metabolites, was carried out through a hemolytic activity test following the methodology cited by Zhen [24] with some modifications as follows: the negative control was phosphate-buffered saline (PBS) (pH 7.2) treated erythrocytes (this buffer protects the erythrocytes against lysis), and the positive control was a solution of 0.1% of Triton-X-100 (Sigma, Milwaukee, WI, USA), a surfactant reagent that is well known to cause cell lysis. The solution was prepared according to the manufacturer’s recommendations). Spectrophotometric analysis was used to measure the absorbance of free hemoglobin (545 nm) in a Thermo Genesys 10S UV/VIS spectrophotometer. The hemolytic activity was reported as percentage of hemolysis (% Hem) evaluated using the following equation:

A − A %Hem = M CN ∗ 100 (1) ACP − ACN where: A = absorbance, M = sample, CN = negative control, CP = positive control. The results were plotted to obtain the hemolytic activity curve (%) vs. concentration of yeast extract.

2.4. Statistical Analysis The experimental design is based on the factorial method 23, which consists of two independent variables: MB concentration and the growth temperature of the removal process with a study of three levels for each variable according to the values reported in Table1. Water 2019, 11, 282 4 of 13

Table 1. Levels and variables involved in the study. Water 2019, 11, 282 4 of 12 Variable Factor Low Level Medium Level High Level Table 1. Levels and variables involved in the study. MB * concentration (ppm) 50 100 200 TemperatureVariable (◦C) Factor Low Level 25 Medium Level 30 High Level 35 MB* concentration (ppm)* MB: Methylene 50 blue. 100 200 Temperature (°C) 25 30 35 In order to evaluate the interactions*MB: of the Methylene variables blue. in relation to the MB bioremediation, the experimental data obtained were processed using the analysis of variance (ANOVA). For the ANOVA study, weIn used order Microsoft to evaluate Excel the interactions statistics. of This the methodologyvariables in relation examines to the dataMB bioremediation, from repeated the assays duringexperimental which both data factors obtained and levelswere areprocessed varied. using the analysis of variance (ANOVA). For the ANOVA study, we used Microsoft Excel statistics. This methodology examines data from repeated 3. Resultsassays andduring Discussions which both factors and levels are varied.

3.1. Effect3. Results of MB and Concentration Discussions and the Growth Temperature on the Removal Process

3.1.The Effect results of MB of theConcentration removal and experiments the Growth areTemperature presented on the in Removal Figure1 Process. Furthermore, in AppendixA (Figure A1The) weresults show of the the removal photographic experiments tracking are presented of the biodegradation in Figure 1. Furthermore, test of MB in Appendix dye for 48 A h (50 ◦ ppm(Figure and 30 A1)C). we In show all cases the photographic examined, wetracking determined of the biodegradation that there was test a of reduction, MB dye for depending 48 h (50 ppm on the temperatureand 30 °C). and In concentration all cases examined, combination we determin used;ed however, that there different was a reduction, percentages depending of these on reductions the weretemperature observed. For and high concentration concentrations combination (100 and used; 200 howe ppm)ver, during different the percentages first 12 h, theof these removal reductions percentage was lowerwere observed. (12% and For 3%, high respectively), concentrations compared (100 an tod MB 200 concentration ppm) during ofthe 50 first ppm 12 (45% h, the of dyeremoval removal). The initialpercentage MB concentrationwas lower (12% playsand 3%, a determinantrespectively), compared role for optimal to MB concentration removal, since of 50 the ppm increase (45% of in the concentrationdye removal). of the The dye initial decreases MB concentration the rate of plays degradation, a determinant probably role for due optimal to the removal, toxic effect since of the the dye on theincrease organism in the [ 25concentration,26]. In addition, of the dye high decreases concentrations the rate of of degradation, dyes cause probably inhibition due of tothe the metabolictoxic effect of the dye on the organism [25,26]. In addition, high concentrations of dyes cause inhibition of processes of microorganisms [27]. Nevertheless, the toxic effect could be modified (i.e., decreased) if the metabolic processes of microorganisms [27]. Nevertheless, the toxic effect could be modified (i.e., moredecreased) yeast is added if more to yeast the solution, is added asto athe result solution, of the as constant a result of ratio thebetween constant theratio MB between concentration the MB and the yeastconcentration content. and the yeast content.

FigureFigure 1. Removal 1. Removal percentages percentages of of MB MB with with initialinitial concentration concentration of of(a)( a50) 50parts parts per permillion million (ppm); (ppm); (b) (b) 100 ppm;100 ppm; (c) 200 (c) ppm200 ppm by exposition by exposition to yeast to yeastG. geotrichum G. geotrichumKL20A KL20A atthree at three different different temperatures temperatures during the 48during h. the 48 h.

Water 2019, 11, 282 5 of 13

Previously, it has been determined that decolorization of a mixture of dyes by G. geotrichum was greatly affected by the addition of various carbon and nitrogen sources, instead of the yeast load [23]. Also, it has been reported that carbon and nitrogen sources have an important influence on the extent of discoloration using microorganisms. Different metabolic characteristics in the microorganisms lead to differences in the uptake of sources, and this also will have an effect on the azo dye discoloration [28–30]. On the other hand, dyes are generally deficient in carbon, and biodegradation without an extra carbon source would be very difficult. For that reason, we used YPD-broth to support cell growth, as evidenced by the hemocytometer count. Carbon sources have two purposes: as sources of energy for the growth of the microorganisms and as electron donors, which are necessary to break bonds [31]. All of these observations suggest that in the future, it will be very important to vary carbon and nitrogen sources to be able to study the effect of the discoloration of different dyes, as well as the effect of the dye structure. However, we found that based on the conditions used in this study, there was an initial efficient load of yeast for a discoloration higher than 70%. Additionally, Figure1 shows that the best biodegradation results are obtained at 35 ◦C (71.5% +/− 0.8%). This result may be correlated with two effects. Firstly, to the optimum temperature for the enzymatic activity in many cells, which is normally placed between 35 and 40 ◦C. Secondly, the result could be related to the mass transfer rate of the dye, since at higher temperatures the viscosity of the solution containing the dye is decreased, increasing the diffusion rate of the dye molecules through the boundary of the outer layer, while at low temperatures this process is slower, decreasing the removal percentage [32]. All the results shown in Figure1 verify that under the above-mentioned conditions, the yeast G. geotrichum presents bioremediation properties towards MB dye. In order to statistically confirm the observed results, a quantitative study was carried out to determine the respective influence of each independent parameter on the response variable (percentage removal). For this study, the analysis of variance of two factors with three replicates per level was used. The combined effects and the percentage of removal (%) obtained from Figure1 are listed in Table2.

Table 2. Effect of the initial dye concentration variable and temperature on the percentage (%) in MB bioremediation from data of Figure1.

Concentration MB (ppm) 25 ◦C 30 ◦C 35 ◦C 9.1 1 62.0 58.9 200 23.7 71.4 56.1 6.2 57.0 44.3 27.7 27.7 26.5 100 32.4 32.4 27.4 35.1 35.1 26.7 58.2 70.7 66.2 50 63.6 64.7 71.7 63.4 62.8 76.6 1 This value corresponds to methylene blue (MB) % bioremoval after 48 h to exposition to G. geotrichum KL20A.

We used ANOVA assay to study the effect of each factor (MB concentration and growth temperature), and the F-distribution (F) was used to determine the statistical effect of each factor. If F Value > F critical Value, then the studied parameter had a statistically significant effect on removal percentage. Table3 lists the ANOVA results obtained from data of Figure1; theoretical aspects can be found in reference [33]. Water 2019, 11, 282 6 of 13 Water 2019, 11, 282 6 of 12 removal conditionsTable 3.wereResults achieved of analysis for a of load variance of 50 (ANOVA)ppm of MB obtained dye and from a temperature data of Figure 1of. 35 °C, which does not deactivate the enzymaticSum of systemDegrees of of the yeasAveraget, reaching of a percentage of 71.5% removal of the Origin of Variations F-Value Probability F-Critical Value MB. Squares Freedom Squares Sample * 6132.4 2 3066.2 98.6 2.0 × 10−10 3.5 Level **Table 3. Results 1618.5 of analysis of 2 variance (ANO 809.3VA) obtained 26.0 from data4.9 of× Figure10−6 1. 3.5 Interaction *** 2586.1 4 646.5 20.8 1.5 × 10−6 2.9 Origin of Sum of Degrees of Average of F-Critical * Factor: This parameter determines if each factor (temperature and BMF-Value concentration) Probability had a statistically significant effectVariations on removal percentage;Squares ** Level:Freedom This parameter Squares determines if each level (25, 30, and 35 ◦C for temperature)Value andSample (50, 100, * and 250 6132.4ppm for BM concentration) 2 had a 3066.2 statistically significant 98.6 effect 2.0 on × removal 10−10 percentage;3.5 *** Interaction: This parameter determines if the interaction between two factors (temperature and MB concentration) hadLevel a statistically ** significant 1618.5 effect on removal 2 percentage. 809.3 26.0 4.9 × 10−6 3.5 Interaction *** 2586.1 4 646.5 20.8 1.5 × 10−6 2.9 *The Factor: results This indicate parameter that determin in all caseses if theeach factor factor F was(temperature significantly and higherBM concentration) than the critical had a value, indicatingstatistically that bothsignificant factors effect studied on removal (temperature percentage and; ** MB Level: concentration) This parameter and determines the levels if of each variation level for each(25, factor 30, and had 35 a °C statistically for temperature) significant and (50, effect 100, onand the 250 results ppm for of BM the concentration) biodegradation had tests.a statistically The results validatedsignificant the observations effect on removal made percentage; in the previous *** Interact sectionsion: This and parameter it is confirmed determines that the if the optimum interaction removal conditionsbetween were two achievedfactors (temperature for a load ofand 50 ppmMB concentration) of MB dye and had a temperaturea statistically ofsignificant 35 ◦C, which effect doeson not deactivateremoval the percentage. enzymatic system of the yeast, reaching a percentage of 71.5% removal of the MB.

3.2.3.2. Effect Effect of of pH pH in in the the Removal Removal Process Process OnceOnce the the optimum optimum conditions conditions of of operation operation for for th thee bioremoval bioremoval process process were were established established (50 (50 ppm, ppm, 3535 °C),◦C), we we carried carried out a study of thethe pHpH mediummedium effecteffect underunder these these conditions. conditions. Three Three pH pH values values (5, (5, 7, 7,and and 9) were9) were evaluated. evaluated. Figure Figure2 shows 2 theshows effects the of theeffects pH basedof the on pH the based percentage on the of biodegradation.percentage of biodegradation.As shown, the pH As is shown, a critical the factor pH is for a MBcritical biodegradation. factor for MB It biodegradation. has been suggested It has that been microbial suggested cells thatare significantlymicrobial cells affected are significant by the pHly ofaffected their immediate by the pH environmentof their immediate because environment they apparently because have they no apparentlymechanism have for adjusting no mechanism their internal for adjusting pH [34 their]. internal pH [34].

FigureFigure 2. 2. EffectEffect of of the the pH pH on on the the biodegradation biodegradation of of methylene methylene blue blue (MB) (MB) dye dye at at the the initial initial MB MB ◦ concentrationconcentration (50 (50 ppm), ppm), and and at at the the temperature temperature of of 35 35 °C.C.

TheThe pH pH of of dyes dyes discharged discharged in in wastewater wastewater varies varies greatly greatly as as a a result result of of their their pH pH dependent dependent nature. nature. ItIt is is well well known known that that the the interaction interaction between between sorb sorbateate and and sorbent sorbent is is also also affected affected by by the the pH pH of of an an aqueousaqueous mediummedium inin two two ways: ways: firstly, firstly, dyes dyes are complexare complex aromatic aromatic organic organic compounds compounds having differenthaving differentfunctional functional groups and groups different and ionizationdifferent ionization potentials potentials at varying at pH, varying which pH, generates which a generates net charge a thatnet chargedepends that on depends the pH ofon the the solution. pH of the Secondly, solution. the Se surfacecondly, ofthe the surface biosorbent of the consists biosorbent of biopolymers consists of biopolymerswith many functional with many groups, functional so the groups, net charge so the on net biosorbent charge on is alsobiosorbent pH dependent. is also pH Therefore, dependent. the Therefore,interaction the between interaction dye molecules between dye and molecules biosorbent and is a biosorbent combined is result a combined of charges result on dyeof charges molecules on dyeand molecules the surface and of the the biosorbent surface of [the35]. biosorbent With an increase [35]. With in pH, an theincrease net negative in pH, the charge net ofnegative the biosorbent charge of the biosorbent increases due to deprotonation of different functional groups [35–37]. However, it has been previously reported that G. geotrichum can decolorize Brilliant blue G dye at a pH between

Water 2019, 11, 282 7 of 13 increases due to deprotonation of different functional groups [35–37]. However, it has been previously reported that G. geotrichum can decolorize Brilliant blue G dye at a pH between 5 to 9, while at pH 3 it has a very low activity, probably due to a loss of the active conformation in the enzymes at low pH (protonation of functional groups by hydrogen ions, losing their activity) [38]. Adsorption and enzymatic activity are dependent on the pH [39]. It has been evidenced that the extent of discoloration is influenced by the pH of the media, as well as that the pH affects the color of the solution and the solubility of the dye [40]. For example, Aspergillus fumigatus decolorates 90% of a real textile effluent containing reactive textile dyes including Reactive Black RC, Reactive Yellow HF2-GL, Reactive Blue BGFN, Reactive Black B-150 and Reactive Red A-6BF at values of 3, 78% at pH 5 and 55% at pH 8 [41]. For these reasons, we selected the pH range between 5 and 9 for the study of the optimal pH solution. At pH 7 a higher bioremoval percentage (71.5%) is reached than at pH 5 and 9 (6% and 12%), respectively. These results are in agreement with other reports; for example, discoloration of mixtures of structurally different dyes at pH 7 and at 30 ◦C in 24 h by action of G. geotrichum MTCC 1360 was reported by Waghmode et al. [23]. Furthermore, these authors reported 87% discoloration of the azo Rubine GFL dye (50 ppm) at 30 ◦C, and pH of 7 in 96 h using G. geotrichum MTCC 1360, (3.0 g malt extract, 3.0 g yeast extract, 5.0 g peptone, and 10.0 g glucose, per liter) [42].

3.3. Kinetic of the Bioremoval Process In order to simulate experimental results of MB kinetic removal, we used a pseudo-first-order model according to Equation (2): dC r = − = k [C] (2) dt t −1 Here, r is the rate of dye removal, kt is the constant rate of the process (h ), [C] is the MB concentration (ppm). Integration of (1) yields:

−kt∗t [C] = [C]oe (3)   [C]t ln = −kt∗t (4) [C]o Figure3a shows the results of the kinetic study and the percentages of removal of MB with an initial concentration of 50 ppm by action of the yeast G. geotrichum KL20A at 35 ◦C. A steep slope is observed for the first 24 h, indicating a faster rate in the removal, while the slope changes to a constant value close to the maximum removal. It is known that the removal is faster at low concentrations of the dye [39], in this case MB, thus in this investigation we used the following conditions for the removal of the MB dye: a temperature of 35 ◦C and a MB concentration of 50 ppm, using a cell concentration of 3.2 × 107 cells/mL of the yeast. Reports in the literature suggest that the several biological species that are studied from the physicochemical point of view follow a pseudo-first order model [43]. Therefore, using such a model, the regression analysis was performed (Figure3) and the process rate constant was obtained. Figure3b shows the results after utilizing the kinetic model (Equation (4)). The value obtained for the kinetic constant of the MB removal process by G. geotrichum KL20A at 35 ◦C was 2.2 × 10−2 h−1 corresponding to a half time for biotransformation of 31.2 h. This value of the kinetic constant is considerably higher than that obtained for other species for the degradation of pollutants: −3 −1 (a) the degradation of polyethoxylates by bacteria, which reported a kt value of 7.2 × 10 h a half time for biotransformation of 96 h [44]; (b) the removal of disperse blue with Klebsiella sp., yields a kt value of 1.0 × 10−3 h−1 for a reaction time of 160 h [45]. Water 2019, 11, 282 8 of 13 Water 2019, 11, 282 8 of 12

FigureFigure 3. ( 3.a) ( Kineticsa) Kinetics of bioremediationof bioremediation of methyleneof methylene blue blue (MB) (MB) at anat an initial initial concentration concentration of 50of ppm50 ppm perper action action of theof the yeast yeastG. geotrichumG. geotrichumKL20A KL20A at35 at ◦35C. °C. (b) ( Modelb) Model L-H L-H on on kinetic kinetic data data of MBof MB removal removal for for ◦ thethe yeast yeast at anat initialan initial concentration concentration of 50of ppm,50 ppm, temperature temperature at 35 at 35C and°C and pH pH 7.0. 7.0.

3.4. Citotoxicity Test 3.4. Citotoxicity Test The cytotoxicity test based on the hemolytic reaction is a highly reliable test in the determination of The cytotoxicity test based on the hemolytic reaction is a highly reliable test in the determination the cytotoxicity of extracts that, in one way or another, may come into contact with human beings [46]. of the cytotoxicity of extracts that, in one way or another, may come into contact with human beings It can be observed in Figure4 that the percentage of hemolysis of the final extracts obtained after the [46]. It can be observed in Figure 4 that the percentage of hemolysis of the final extracts obtained after discoloration of MB dye experiments was lower than what was obtained for MB solutions, indicating the discoloration of MB dye experiments was lower than what was obtained for MB solutions, a lower cytotoxicity property for the extracts as compared to the MB solutions. It is important to indicating a lower cytotoxicity property for the extracts as compared to the MB solutions. It is assess cytotoxicity of the final extracts, since the metabolites produced from dye degradation are, in important to assess cytotoxicity of the final extracts, since the metabolites produced from dye manydegradation cases, more are, toxic in many than cases, the parent more dye. toxic For than example, the parent the dye. products For example, of the oxidation the products of indigo of the blue via electro-incineration and chemical coagulation with Al2(SO4)3 are more toxic than the parent oxidation of indigo blue via electro-incineration and chemical coagulation with Al2(SO4)3 are more dyetoxic [31]. than It is the important parent dye to note [31]. that It is the important extract withoutto note that purification the extract procedures without purification (to remove procedures medium and(to yeast) remove is muchmedium less and cytotoxic yeast) is (less much percentage less cytotoxi of hemolysis)c (less percentage compared of hemolysis) to the original compared solution to the of theoriginal MB. solution The metabolites of the MB. produced The metabolites from dye produc degradationed from are, dye in degradation many cases, are, more in many toxic cases, than themore parenttoxicdye than [39 the]. parent Several dye azo [39]. dyes Several and the azo amines dyes and from the their amines degradation from their have degradation shown mutagenic have shown responsesmutagenic in Salmonella responses in and Salmonella mammalian and assay mammalian systems, a andssay theirsystems, toxicity and depends their toxicity on the depends nature andon the positionnature of and the position substituents of the in thesubstituents molecule [in47 the]. Therefore, molecule it[47]. is very Therefore, important it is for very any important bioremediation for any technologybioremediation to evaluate technology the toxicity to evaluate of the the pollutants toxicity of and the metabolites pollutants and formed metabolites after dye formed degradation after dye indegradation order to study in order the feasibility to study the of thefeasibility method of [ 48the]. method Toxicity [48]. should Toxicity be evaluated should be usingevaluated various using methodologiesvarious methodologies with respect with to phytotoxicity,respect to phytotoxicit ecotoxicity,y, ecotoxicity, genotoxicity, genotoxicity, mutagenicity, mutagenicity, acute toxicity, acute microbialtoxicity, toxicity, microbial and toxicity, toxicity onand invertebrates. toxicity on invert Normally,ebrates. the testNormally, chosen the corresponds test chosen to thosecorresponds that are to morethose economic that are or more accessible economic in terms or accessible of the time in terms and feasibility of the time to and test feasibility the samples to [test39]. the However, samples we [39]. didHowever, not investigate we did the not mechanism investigate of dyethe mechanism degradation, of which dye degradation, will be the subject which of will a forthcoming be the subject study, of a withforthcoming the identification study, ofwith some the metabolitesidentification produced of some after metabolites the biodegradation produced after process the andbiodegradation different toxicityprocess assays, and includingdifferent thetoxicity oxidative assays, stress including response. the Nevertheless, oxidative stress the cytotoxicityresponse. Nevertheless, analysis of the the finalcytotoxicity extracts resulting analysis fromof the the final degradation extracts resultin processg from (without the degradation any further process purification (without or extraction any further process)purification demonstrated or extraction a substantial process) reduction demonstrated of the a cytotoxicitysubstantial reduction to human of erythrocytes, the cytotoxicity a result to human that deserveserythrocytes, a deeper a investigation,result that deserves since normally a deeper itinvestigation, is the phytotoxicity since normally of the extracts it is the that phytotoxicity is evaluated. of Phytotoxicitythe extracts ofthat the is finalevaluated. extracts Phytotoxicity has been observed of the final to decreaseextracts has for beenG. geotrichum observedbioremediation to decrease for G. processes.geotrichum For bioremediation example, a consortium-GB processes. For example, containing a co twonsortium-GB microorganisms, containingG. two geotrichum microorganisms,MTCC 1360G. andgeotrichum Bacillus MTCC sp. VUS, 1360 was and able Bacillus to degrade sp. theVUS, sulfur-containing was able to degrade dye Brilliant the sulfur-containing Blue G optimally dye ◦ atBrilliant pH 9 and Blue at G 50 optimallyC, and theat pH phytotoxicity 9 and at 50 °C, test and revealed the phytotoxicity the nontoxic test naturerevealed of the the nontoxic metabolites nature resultingof the metabolites from the degradation resulting from process. the degradation In another study, process.G. geotrichumIn another MTCCstudy, 1360,G. geotrichum showed MTCC 88% ADMI1360, (American showed 88% Dye ManufacturingADMI (American Institute) Dye removalManufact ofuring the mixtureInstitute) of structurallyremoval of differentthe mixture dyes of structurally different dyes (Remazol red, Golden yellow HER, Rubine GFL, Scarlet RR, Methyl red, Brown 3 REL, Brilliant blue) (70 mg L−1) within 24 h at 30 °C and pH 7.0 under shaking conditions

Water 2019, 11, 282 9 of 13

(RemazolWater 2019, 11 red,, 282 Golden yellow HER, Rubine GFL, Scarlet RR, Methyl red, Brown 3 REL, Brilliant9 blue)of 12 (70 mg L−1) within 24 h at 30 ◦C and pH 7.0 under shaking conditions (120 rpm). The phytotoxicity (120 rpm). The phytotoxicity study indicated the conversion of the complex dye molecules into study indicated the conversion of the complex dye molecules into simpler oxidizable products having simpler oxidizable products having a less toxic nature. a less toxic nature.

Figure 4. Comparison of hemolytic activity (%) to: ( a) methylene methylene blue (MB), ( b) yeast yeast extracts extracts obtained from MBMB removalremoval tests,tests, ((c) negativenegative controlcontrol (phosphate-buffer-treated(phosphate-buffer-treated erythrocytes, PBS) and ( d) positive control (Triton-X-100).(Triton-X-100).

4. Conclusions The removal of the MB dyedye atat differentdifferent initialinitial loadsloads (50(50 ppm,ppm, 100 ppm,ppm, and 200 ppm) was 7 evaluated usingusing aa yeastyeast strainstrainG. G. geotrichumgeotrichumKL20A KL20A with with an an initial initial concentration concentration of of 3.2 3.2× ×10 107 cells/mLcells/mL ◦ for aa reactionreaction timetime of of 48 48 h h at at 25, 25, 30, 30, and and 35 35C. °C. Results Results indicated indicated that that the the increasing increasing temperature temperature in the in studythe study range range had ahad positive a positive effect effect on the on removal the remo process;val process; 76.6% removal76.6% removal was obtained was obtained at the highest at the ◦ temperaturehighest temperature of 35 C, of with 35 a°C, dye with concentration a dye concen of 50tration ppm. Onof 50 the ppm. other On hand, thethe other MB hand, concentration the MB hadconcentration an adverse had effect an onadverse the removal effect on process. the remo Theval highest process. removal The highest percentage removal was percentage obtained at was the lowestobtained MB at concentration, the lowest MB as otherconcentration, authors haveas ot alreadyher authors demonstrated; have already this isdemonstrated; presumed to bethis due is topresumed intoxication to be of due the to microorganism intoxication of oncethe microorga the concentrationnism once increases. the concentration The study increases. of kinetics The allowed study usof kinetics to calculate allowed the valueus to ofcalculate the kinetic the value constant of the of thekinetic MB constant removal of process the MB by removalG. geotrichum processKL20A by G. −2 −1 atgeotrichum optimal KL20A conditions. at optimal The kinetic conditions. study The yielded kinetic a rate study constant yielded 2.2 a rate× 10 constanth and 2.2 × a 10 half−2 h time−1 and for a biotransformationhalf time for biotransformation of 31.2 h. The of hemolytic 31.2 h. The reaction hemolytic test is consideredreaction test acceptable is considered and favorable,acceptable since and thefavorable, hemolytic since activity the hemolytic (% hem) activity was the (% lowest hem) percentagewas the lowest reported percentage for the reported positive for control the positive and for thecontrol initial and MB. for It the is importantinitial MB. to It note is important that the final to note sample that ofthe MB final obtained sample after of MB the obtained yeast treatment after the is muchyeast treatment less cytotoxic is much (less% less hemolysis) cytotoxic compared (less% hemoly to thesis) initial compared MB solution to the without initial MB any solution yeast treatment, without whichany yeast demonstrated treatment, thatwhichG. demonstrated geotrichum KL20 that was G. effective geotrichum to removeKL20 was MB effective with a substantial to removereduction MB with ofa substantial the cytotoxicity reduction to human of the erythrocytes. cytotoxicity to human erythrocytes. All of the results confirmedconfirmed that G. geotrichum KL20 can be used for the removal of effluents effluents with MB dyedye loads,loads, butbut it it would would be be valuable valuable in in future future studies studies to compareto compare the concentrationsthe concentrations used used here withhere thewith concentrations the concentrations of MB of in MB real in streams real streams of wastewater. of wastewater.

Author Contributions: Conceptualization, M.C., C.D.G.-T., W.V. and C.C.-L.; methodology, M.C., C.D.G.-T. Author Contributions: Conceptualization, M.C.,C.G.-T.,W.V. and C.C.-L.; methodology, M.C.,C.G.-T. and W.V.; and W.V.; validation, M.C., C.D.G.-T. and W.V.; formal analysis, M.C.,C.D.G.-T. and W.V.; M.C.,C.D.G.-T. and W.V.;validation, C.D.G.-T.,W.V. M.C.,C.G.-T. and and C.C.-L.; W.V.; data formal curation, analysis, M.C.,C.D.G.-T. M.C.,C.G.-T. and W.V.; writing—originalM.C.,C.G.-T. and W.V.; draft C.G.-T.,W.V. preparation, M.C.,C.D.G.-T.and C.C.-L.; data and curation, W.V.; writing—reviewM.C.,C.G.-T. and andW.V.; editing, writing—original M.C.,C.D.G.-T.,W.V. draft preparatio and C.C.-L.;n, M.C.,C.G.-T. visualization, and W.V.; X.X.; supervision,writing—review C.D.G.-T. and editing, and W.V.; M.C.,C.G.-T.,W.V. project administration, and C.C.-L.; C.D.G.-T. visualization, and W.V.; X.X.; funding supervision, acquisition, C.G.-T. C.D.G.-T.,W.V. and W.V.; andproject C.C.-L. administration, C.G.-T. and W.V.; funding acquisition, C.G.-T.,W.V. and C.C.-L. Funding: This research was funded by Universidad del Atlántico, First internal call for support for the Funding: This research was funded by Universidad del Atlántico, First internal call for support for the development of degree work in training research-undergraduate and graduate level 2018. development of degree work in training research-undergraduate and graduate level 2018.

Acknowledgments: The authors thank to the Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo for the donation of the strain of Galactomyces geotrichum KL20 used in the present study.

Water 2019, 11, 282 10 of 13

Acknowledgments: The authors thank to the Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo for the donation of the strain of Galactomyces geotrichum KL20 used in the presentWater 2019 study., 11, 282 10 of 12 Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix A

Figure A1.A1. TestsTests of of biodegradation biodegradation of MBof MB dye dye bythe by actionthe action of the of yeast the Galactomycesyeast Galactomyces geotrichum geotrichumKL20A. (KL20A.a) Time (a zero) Time hours, zero ( bhours,) Time (b 12) Time h, (c )12 Time h, (c 24) Time h, ( d24) Timeh, (d) 48 Time h, (MB 48 h, concentration (MB concentration was 50 was ppm 50 andppm temperature and temperature was 30was◦C). 30 °C). The The images images on theon the left left correspond correspond to to replays replays (× (×3)3) of ofthe the degradationdegradation experiments. TheThe images images on on the the right right correspond correspond to the to controls the controls used inused order in of order yeast, of culture yeast, medium culture (YPD-broth).medium (YPD-broth).

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